Somatic and Flagellar Immunofluorescence

Transcription

1 JOURNAL OF BACTERIOLOGY, Oct., 966 Copyright 966 American Society for Microbiology Vol. 92, No. Prinited in U.S.A. Somatic and Flagellar Immunofluorescence of Salmonella' W. J. CALDWELL, C. S. STULBERG, AND W. D. PETERSON, JR. The Child Research Center of Michigan and Department of Microbiology, Wayne State University School of Medicine, Detroit, Michigan Received for publication 5 June 966 ABSTRACT CALDWELL, W. J. (The Child Research Center of Michigan, Detroit, Mich.), C. S. STULBERG, AND W. D. PETERSON, JR. Somatic and flagellar immunofluorescence of Salmonella. J. Bacteriol. 92: Labeled globulin fractions of flagellar (H) antisera, prepared against 2 frequently occurring Salmonella serotypes belonging to five major somatic () groups, were characterized for and H immunofluorescence and for and H agglutinin titers against 2 serotypes. The feasibility of immunofluorescent identification of both somatic and flagellar antigens was enhanced by staining formaldehyde-treated organisms in suspension. Relationships between homologous, partial, and unrelated antigen-antibody systems were then analyzed, and a high degree of correlation was shown between the results obtained by the two serological procedures. Flagellar staining was highly specific, and was bright, faint, or inapparent, depending on the relationship between the antigenantibody systems involved. Somatic staining was also specific, but somewhat more difficult to interpret, because cells in the same preparation might exhibit a mixture of bright, faint, or no fluorescent intensities. Correlation was shown between the percentage of brightly staining cells found in these preparations and the agglutination titers of the comparable antigen-antibody systems. The phenomenon of a "percentage" reaction was unexplained. Absorption studies further confirmed the specificity of reactions. The techniques developed were applied to surveillance of several mouse colonies for the presence of Salmonella. Broth cultures of fecal specimens were treated with formaldehyde and stained in suspension with "polyvalent" labeled antibody reagents. Agreement was found in 97.6% of the instances between results obtained by immunofluorescence and cultural methods. In addition, preliminary evidence indicated the feasibility of presumptive serotyping of Salmonella isolates by immunofluorescence. The first application of the well-known uses of the fluorescent-antibody technique to the antigenically complex Salmonella was made by Thomason, Cherry, and Moody (). They showed that somatic (), flagellar (H), or Vi fluorescent antibody that specifically stained the corresponding antigens of Salmonella typhosa in pure culture could be prepared. However, the nonspecific staining of other organisms present in fecal smears markedly hindered the diagnostic application of the method (9), although specificity was conferred in instances where Vi antigens were present (). Several modifications were developed and I Presented in part at the 6th Annual Meeting of the American Society for Microbiology, Washington, D.C., May 96. adopted in this study that differed from those reported above (, 9, ). Most importantly, fluorescein-labeled H antisera, which were characterized for both and H antibody content, were employed, and staining was carried out by mixing formaldehyde-treated cells in suspension with labeled antibody. These differences permitted observation of both and H reactions in the same preparation. This methodology has been applied with success to the epidemiological and diagnostic study of a hospital Salmonella outbreak previously reported from this laboratory (8). The present report is concerned primarily with the specificity and sensitivity of immunofluorescence for somatic and flagellar antigens, with the agglutination reaction as the basis for 77 Downloaded from on November 2, 28 by guest

2 78 CALDWELL, STULBERG, AND PETERSON J. BACTERIOL. comparison. Further applications methodology are also described. of the MATERIALS AND METHODS Salmonella serotypes. Standard strains of Salmonella were kindly provided by the Salmonella Typing Station, Michigan Department of Public Health, Lansing, Mich. The 2 strains employed included representatives of the most frequently encountered serotypes; they are listed with their antigenic formulations in Table. The majority of strains were monophasic with respect to flagellar antigens. TABLE. Preparation ofantigens. Antigens were prepared for rabbit immunization essentially according to the method of Edwards and Ewing (2). Stock culture strains were first grown in Veal Infusion Broth (Difco), and then were plated on Veal Infusion Agar (Difco) from which typical colonies were picked and serially cultivated in semisolid medium of Edwards and Bruner (BBL) for the selection of actively motile strains. Subsequently, these were grown in Veal Infusion Broth for 6 hr, were tested for purity, and were inactivated with.5% formaldehyde. Concentrations approximated 9 cells per milliliter. Salmonella serotypes employed as antigens for preparationt of anitisera, fluorescein-labeled antibodies, and serological reactions Groupa Salmonella species and strain designationb Somatic () antigens A B C D E F G H S. paratyphi A 5c S. paratyphi B var. javac S. paratyphi B N-c S. typhimurium 66 S. typhimurium 6 S. typhimurium 95 S. typhimurium CDA78C S. typhimurium 7 S. reading 2682c S. typhimuriuim var. copenhagen 6 S. typhimurium var. copenhagen 659 S. derby c S. esseni 7c S. abortivoequine beta 22c S. schleissheim alpha c S. thompson 968c S. thompson var. berlin 2988c S. montevideo 62c S. oranienburg 987c S. newport 56 S. typhosa H 9Wc S. enteritidis 89c S. dublin c S. gallinarum 6 S. strasbourgc S. anatum 29c S. newington C S. simsbury 77c S. rubislaw S. worthington 6c S. madelia 6C S. carrau,2,2,5,2,5,2,,5,2,5,2,5,2,5,2,5,2,5,2,,2,2,2,2,2,2,27 6,8 9,2 9,2 9,2 9,2 (9),6,,5,,9,,2 (),6,, 6,,2 Flagellar (H) antigens Phase Phase 2 a b (i) i i if,g g,m b k g,m,s m,t a Kauffmann-White schema. b Standard strains obtained from the Salmonella Typing Station, Michigan Department of Public Health, Lansing, Mich. c Labeled antisera prepared against these strains. d g,m g,p d r y y,2,2,2,2,2,2 e,n,x,5,2,7,6,6 Z27 e,n,x,7 Downloaded from on November 2, 28 by guest

3 VOL. 92, 966 IMMUNOFLUORESCENCE OF SALMONELLA 79 Antigens for use in H and agglutination reactions were prepared in the same manner as the immunizing antigens, except that antigens were heated at C for 2 hr before the addition of.5% formaldehyde. Absorbing antigens, similarly prepared, were grown on Veal Infusion Agar for 6 hr. A concentration of approximately 2 X formaldehyde-treated, washed cells was used for each of two absorptions of labeled globulins diluted :. Antisera and agglutination tests. H antisera were prepared by inoculating two rabbits per serotype intravenously with three injections per week at increasing doses [.,.5,.,.5, 2. (X) ml] of the immunizing antigens. Rabbits were bled 7 days after the last injection, and the collected sera were stored frozen (- C) until tested or labeled. A preinjection serum was also tested from each animal. The H and antibody titers of each of the antisera were determined by tube agglutination tests performed at 5 C. Readings of agglutination titers were made after 2 hr of incubation with H antigens and after 8 hr of incubation with antigens. Fluorescent-antibody procedure. Globulin fractions of the antisera were prepared by precipitation in 8% sodium sulfate ( volume of serum per 2 volumes of 27% sodium sulfate) at 7 C for 8 hr. After dialysis, the globulin solutions, adjusted to ph 9., were conjugated with fluorescein isothiocyanate (FITC) by methods previously reported (7). The globulin solutions were added to a dry mixture of part of FITC (. mg of FITC per mg of protein) and 9 parts of cellulose powder (Celite), and were mixed for 5 min. Celite was removed by centrifugation, and the unbound dye, by gel filtration (Sephadex G medium). All conjugated globulin preparations were stored at - C. For fluorescent-antibody reactions, cells were cultured simultaneously from the same source as that used for agglutination tests. Broth cultures were centrifuged for 2 min at 86 X g, and the sediments were suspended in % formaldehyde for min. The formaldehyde-treated cells were then centrifuged and resuspended in one-half their original volume in.2 M phosphate buffer (ph 8.). The organisms were stained by adding. ml of the formaldehyde-treated cell suspension to. ml of labeled antibody (final dilution, :2 in phosphate buffer). The mixture was then agitated for min on a rotary shaker at room temperature. After the staining period, 2. ml of buffer solution was added to the suspension with subsequent centrifugation at 86 X g for 2 min. After two additional washings, the final sediment was left in approximately. ml of buffer. About. ml of this suspension was placed within an inscribed area on a slide, was air-dried, was mounted under a no. cover slip in a drop of buffered glycerol, ph 9. ( part carbonate-bicarbonate buffer, 9 parts glycerol), and then was sealed with lacquer. All preparations were observed by fluorescence microscopy with the use of a BG-2 pass filter and an OG- barrier filter. Fluorescent-antibody identification of Salmonella in specimens. Mouse fecal pellets were collected during a 6-month period from several breeding colonies where one animal per cage was tested. Each fecal specimen was placed in 5 ml of dextrose broth, was refrigerated approximately 8 hr during transit, and was then incubated for 6 hr prior to testing. From these broth cultures, a sample was treated with formaldehyde as described, and immunofluorescence procedures were carried out directly. A labeled polyvalent antiserum pool was prepared with a final dilution of :2 for each of the eight labeled antisera employed. The remaining broth sample was cultured on selective plating media for isolation of lactose-negative colonies which were then identified by standard cultural and serological procedures. Salmonella isolates from infected children were similarily grown in broth culture and were treated with formaldehyde for immunofluorescence determinations. For serotyping of these, Spicer-Edwards polyvalent H antisera were kindly provided by Difco. These antisera were labeled and characterized by and H tube agglutination tests as described above. RESULTS Immunofluorescence staining reactions. Typical immunofluorescence reactions of Salmonella stained with labeled antibody are illustrated in Fig.. Somatic staining was observed as peripheral fluorescence of these organisms (Fig. IA); irregular or spotty staining was rarely seen. Flagella stained as single fluorescent strands (Fig. IA) or as agglutinated masses (Fig. ib). The latter was typical of reactions encountered when labeled antibody was specific for flagellar antigens only. Within a population of cells on a slide, somatic staining varied in intensity. On the basis of approximately fields per slide, the proportion of cells that stained brightly, faintly, or not at all, was estimated from the total number of cells visible by tungsten illumination. Figures IC and D illustrate reactions wherein 95% and 5% of the cells, respectively, stained brightly. In contrast, flagella stained uniformly, and their fluorescence was either bright or faint. The cells shown in Fig. le were illuminated by tungsten light. The same field, illuminated by ultraviolet light, showed specifically stained flagella as bright fluorescent strands in the absence of somatic fluorescence (Fig. F). Because flagella were not visible by tungsten light, estimates of the proportion of cells with stained and unstained flagella could not be made. Characterization and selection of labeled globulin preparations. Somatic and flagellar agglutination titers were determined for each of the 2 conjugates and 2 whole antiserum preparations by reaction with each of the 2 serotypic strains. These agglutinations were grouped according to the antigenic-factor relationships (5) that existed between the antigen-antibody systems employed. They were designated as "homologous" or identical; "similar," or having all Downloaded from on November 2, 28 by guest

4 8 CALDWELL, STULBERG, AND PETERSON J. BACTERIOL. Downloaded from FIG.. Photomicrographs ofpure cultures and isolates ofsalmonella stained with fluorescein-labeled antibodies. Dark fields illuminated by near-ultraviolet or tungsten are shown. In some instances, the fluorescent organisms were purposely overexposed to demonstrate flagellar staining, and, thus, the microorganisms appear to be solidly stained. (A) S. thompson stained with homologous labeled antibody illustrating peripheral somatic fluorescelce andfluorescenice ofa single flagellum. (B) S. indiana isolate stained with labeled Spicer-Edwards pool containing labeled flagellar (but not somatic) antibody for this strain; a fluorescent flagellar mass is shown. (C) S. newport isolate stained with labeled Spicer-Edwards pool ; bright somatic staining of a high percentage of the organisms as well as flagellar staining are illustrated. (D) S. typhimurium stained with labeled S. typhosa antibody; approximately 5%hc of the organisms stained brightly in this partially related system; unstained bacteria are barely visible. (E) Dark-field illuminated by tungsteni light showing all the microorganisms in the preparation. (F) Same field illuminated by ultraviolet showing that only the flagella were fluorescent when flagellar labeled antibodies were homologous with the strain (somatic antibodies were unrelated). somatic in common; "partial," or having one or two somatic or flagellar in common; and "unrelated," or possessing no antigenic in common. When somatic agglutination titers were compared in labeled and unlabeled preparations, reactions falling within a fourfold antibody dilution occurred with 9% of the homologous systems, 8% of similar on November 2, 28 by guest

5 VOL. 92, 966 IMMUNOFLUORESCENCE OF SALMONELLA 8 systems, 8.5% of partial systems, and 96% of unrelated systems. With flagellar reactions, the titers obtained with 95% of the homologous, 86.5% of the partial, and 98% of the unrelated systems were within a fourfold dilution of each other in the labeled and unlabeled preparations. To make the most meaningful interpretation of immunofluorescence reactions, only those conjugates that had titers comparable to that of whole serum, i.e., within a fourfold difference, were used. Somatic immunofluorescence. Table 2 summarizes the somatic immunofluorescence and agglutination reactions carried out with labeled globulin fractions. Of 8 homologous systems studied, all exhibited bright, peripheral, fluorescent staining of 7 to % of the cells (see Fig. IC). The remaining cells, in these preparations, showed faint fluorescence. Agglutination titers ranged from : 6 to : 5,2 with a modal value of :6. Of systems designated similar, reacted in like manner, whereas had somewhat lower agglutination titers corresponding to a lower ( to 6%) level of bright staining. TABLE 2. Comparison of somatic immunofluorescence and agglutiniation reactions of labeled globulins No. of paired Immunofluorescence reactions Agglutination reactions FA and agglutina- systems tionystemsa No. of Organisms < :2 to :6 to :,28 to tested" Intensity systems stained <2 :8 :6 :5,2 Ttl Somatic antigenantibody s.ystem antbodsyte Homologous Total Similar Total Partial Total Unrelated Total 8 9 a FA, fluorescent antibody. Bright Faint Negative Bright Faint Negative Bright Faint Negative Bright Faint Negative to 7 to 8 9to 7 to 8 to 6 9 to 7 to 8 to 6 5 to 5 to >Oto2 9 to 7 to 8 to 6 5 to 5 to >Oto2 5 to >Oto2 7 to 8 to 6 5 to 5 to >Oto Downloaded from on November 2, 28 by guest

6 82 CALDWELL, STULBERG, AND PETERSON J. BACTERIOL. in E.In -o. c a C: I- O) Of the 9 unrelated systems, detectable agglutination titers were recorded for only, whereas fluorescence was noted for 7. Most of the fluorescence reactions involved a small (% or less) percentage of the cells in a preparation. Thus, 5 of 7 systems exhibited bright fluorescence in 2% or less of the cells, whereas 98 of instances showed faint fluorescence of less than 6%. Only two of these instances were accompanied by detectable agglutination reactions. Therefore, fluorescence at the levels indicated might be nonspecific. Bright fluorescence of 5 to % or faint fluorescence of 7 to % was considered to be of borderline significance. These conclusions were emphasized by the cross-reactions of the partially related systems. Bright fluorescence of 7 to % of the cells was generally accompanied by substantial agglutination titers, and bright fluorescence of 5 to %, by low agglutination titers. An intermediate grouping was also apparent, when occasional faint somatic fluorescence of 7 to % was accompanied by little or no agglutination. Fluorescence reactions of lesser degree again appeared to be nonspecific in denoting relationship, as judged by the agglutination reactions observed. With these criteria applied to the 52 reactions where partial relationship was not expressed by detectable agglutination titers, 7 might also be considered negative by immunofluorescence. In contrast, in of the partially related systems, low agglutination titers were found in the absence of significant immunofluorescence. Thus, substantial correlation between somatic agglutination titers and immunofluorescence was apparent when closely related systems were involved, but agreement between the two methods varied when the relationships were minor. A specific example is illustrated in Fig. 2 where unabsorbed labeled antibody for S. abortivoequina (containing somatic,2) was tested against the homologous as well as three partially related strains, S. typhimurium (,5,2), S. typhosa (9,2), and S. paratyphi A (,2,2). Bright fluorescence of 9% of the organisms, together with high agglutination titers, characterized the homologous and closely related somatic of the two group B strains. Fluorescence of a much lower percentage of organisms and correspondingly lower agglutination titers characterized the less closely related strains. The absorptions illustrated in Fig. 2 further confirmed the specificity of the reactions observed with the unabsorbed labeled antibody. Fluorescence reactivity was absorbed more readily than was agglutination; this suggested differences in the sensitivity of the two methods. After absorption, the closer relationship of,2 and,5,2 (group B) strains to each other than to Fluorescein - labeled antibody to S. Gbortus-equi(,/2) Absorbed witl,5,2 _,2,2 Downloaded from on November 2, 28 by guest *reciprocal of final dilution M bright staining faint staining FIG. 2. Comparative somatic fluorescence and agglutination reactions of Salmonella abortivoequina (, 2) labeled antibody before and after absorption with homologous and related somatic antigens.

7 VOL. 92, 966 IMMUNOFLUORESCENCE OF SALMONELLA the,2, 2 (group A) or 9, 2 (group D) strains was as apparent by fluorescence as by agglutination. Interestingly, another antibody prepared specifically against S. typhimurium (,5,2) reacted well by agglutination but gave low percentage reactions ( to %) against,2 antigens, possibly because of the dominance of the 5 factor in that antiserum. Thus, by examination of group B strains with both conjugates,,5,2 serotypes could be readily distinguished from,2 serotypes by their characteristic percentage reactions. Figure 2 also indicates the specificity of lowpercentage bright and high-percentage faint fluorescence reactions. The % bright and the 6% faint fluorescence of 9,2 antigen with,2 antibody was completely absorbed by all but antigen,2,2, as was the accompanying agglutination titer. Similarly, the reactivity against,2,2 antigen could be absorbed, but in both cases the reactivity with,2 and,5,2 antigens was unaffected. Figure illustrates the reactions of a labeled antibody preparation specific for S. typhosa (9,2), which reacts specifically with the homologous antigen and minimally, or not all, with partially related strains. This antibody also provided an example of bright staining of S. typhimurium (,5,2) at the 5% level in the absence of agglutination. The small percentage E(I, r-.ev ca r- c- ~. to v) O- C._) c CD c: reaction was absorbed by all antigens except,2,2. Further, the reaction with,5,2 antigen persisted in the same proportion after colony isolates had been passed through several subcultures, and the reaction was not altered by physiological age of the cultures. Growth of S. typhimurium in specific 9,2 antiserum blocked the reaction, but subsequent subcultures free from serum showed the same percentage reactivity, which demonstrated its specificity. Flagellar immunofluorescence. Comparative immunofluorescence and agglutination flagellar reactions were carried out with labeled globulin fractions as summarized in Table. The fluorescent-antibody observations were made at the same time as the somatic reactions previously described. Nineteen homologous systems were studied, and, in each, the labeled antibodies produced bright staining which occurred as masses of fluorescent flagella. This correlated with a modal agglutination titer of :,2. There were 6 partially related flagellar antigen-antibody systems. Of these, were characterized by bright flagellar fluorescence of many flagella, and high agglutination titers were found. However, six involved more distantly related systems in which only a few single flagella stained and which were consistent with little or no agglutination. Similarly, six faint and eight negative fluorescence reactions could be correlated with Fluorescein - labeled antibody to S t.yphosa (9, /2 ) -Ir-I Absorbed with 8 Downloaded from on November 2, 28 by guest *reciprocal of final dilution bright staining a faint staining FIG.. Comparative somatic fluorescence and agglutination reactions of Salmonella typhosa (9, 2) labeled antibody before and after absorption with homologous and related somatic antigens showing the specificity of the 5% fluorescence reaction with S. typhimurium (,5,2).

8 8 CALDWELL, STULBERG, AND PETERSON J. BACTERIOL. TABLE. Comparison of flagellar immunofluorescence and agglutination reactions of labeled globulins No. of paired IlfllfIunofluorescence Agglutination reactions Flagellar antigen- FA and antibody system agglutination No of :- :2- :2,56- :28- systems testeda Intensity systems < : :6 :,28 :,2 :,96 Homologous 9 Bright 9 5 Faint Negative Partial 6 Bright Faint Negative 6 2 Unrelated 96 Bright 2 2 Faint 2 Negative Totals afa, fluorescent antibody. low or negligible agglutination titers which involved sera that were nonreactive with antigens having minor factor relationships, g,m,s; m,t;, 6;, 7 (2). Two, with no fluorescence, but with higher agglutination titers, were not further verified. The majority of flagellar reactions fell in the unrelated grouping. Of 96 preparations, immunofluorescence was not observed, except for 2 in which only a few brightly stained flagella were seen, and which stained faintly with low agglutination titers. Of 9 reactions negative by immunofluorescence, 9 had insignificant agglutination titers. The high degree of specificity of flagellar reactions was emphasized by agreement of immunofluorescence and agglutination reactions in 558 of 579 instances (96.%). Further evidence of their specificity was obtained by absorption of all reactivity in each of the 9 homologous systems. In addition, the absorption experiments described for somatic antibodies involved the monophasic flagellar for S. abortivoequina (enx), S. typhimurium (,2), S. typhosa (d), and S. paratyphi B (b) indicated in Table. In each instance, homologous absorption completely removed both fluorescent-antibody and agglutinating reactions for flagellar antigens but not the heterologous reactivity. Absorption with somatic antigens did not alter flagellar antibodies. Presumptive identification of Salmonella by immunofluorescence. The foregoing analyses of both somatic and flagellar fluorescent-antibody reactions, together with the successful diagnostic and epidemiological application of a labeled preparation for S. typhimurium (8), suggested that labeled polyvalent-preparations incorporating combined somatic and flagellar antibodies might be employed for presumptive identification or screening purposes. Two types of polyvalent fluorescent antibodies were employed: combinations of labeled preparations (Table ) and a labeled Spicer-Edwards preparation (Table 5). The former was tested for detection of salmonellosis in mice, whereas the latter was used in attempts to identify Salmonella isolates from children. Mouse fecal specimens were obtained and cultured as described. Portions of 6-hr broth cultures were stained with polyvalent labeled antibody, and only cells exhibiting both somatic and flagellar fluorescence were considered positive for Salmonalla. The remainder of the broth cultures were subcultured to MacConkey and SS Agar (Difco) for subsequent isolation and confirmation of lactose-negative colonies as Salmonella. The polyvalent labeled antibody was prepared by pooling antisera against serotypes most likely to be encountered in naturally infected mice. Of a total of 7 specimens, shown in Table, 5 yielded organisms exhibiting both somatic and flagellar fluorescence and, thus, were considered positive for Salmonella. These were confirmed by definitive culture and by presumptive serotyping by conventional methods. The majority (66) of specimens were judged negative by both procedures. Of these, somatic fluorescence alone was found in 67, but, because flagellar reactions were absent, and because most somatic reactions appeared to be due to unidentified coccobacilli, they were judged to be nonspecific. Twenty specimens revealed flagellar staining alone, usually associated with a large fusiform Downloaded from on November 2, 28 by guest

9 VOL. 92, 966 TABLE. Comparison of immunofluorescencea and culture for detection of Salmonella in mouse fecal specimens No. of specimens IMMUNOFLUORESCENCE OF SALMONELLA Results on paired broth specimens Immunofluorescence (-H) positive) Culture positve a Composition of polyvalent labeled-pool: S. typhimurium, (,5,2:i,-); S. paratyphi B, (,5,2: -,,2); S. essen, (,2:gm,-); S. thompson, (:-,,5) and (:k,-); S. oranienburg, (: mt,-); S. anatum, (,: eh,j,6). bacillus having short peritrichous flagella, and these were also judged nonspecific. In an additional specimens, somatic and flagellar fluorescence of organisms morphologically similar to Salmonella was observed, but the lactose-negative colonies isolated were not Salmonella. Only four isolations of Salmonella were obtained from specimens which were negative by immunofluorescence. Of the entire series, it was noteworthy that there was agreement in nearly all instances (97.6%) between cultural and immunofluorescence findings. It was of further interest to determine whether fluorescein labeling of Spicer-Edwards polyvalent antisera could be used for presumptive screening of Salmonella isolates. Although it will be recalled that such H antisera were designed for presumptive typing (by flagellar ) of previously grouped Salmonella, the foregoing data suggested that both flagellar and somatic might be recognized by immunofluorescence, especially if the "percentage" reactions could be utilized. For this purpose, Spicer-Edwards polyvalent pools to (Serological identification of the Salmonella, Difco Laboratories, Detroit) were labeled and applied to formaldehyde-treated broth preparations of seven untyped isolates obtained from infants. The somatic and flagellar staining reactions of these isolates are shown in Table 5. By noting the presence or absence of bright somatic and flagellar staining, it was possible to compile a list of most probable serotypes for each isolate, arranged in a descending order of frequency of isolation from the community over a period of 5 years (information obtained from Division of Epidemiology, Michigan Department of Public Health). The isolates were then typed by the usual procedures, and, in each instance, the 85 definitive serotype was contained in the list of those considered most probable. Of interest were the reactions of isolates and 2, where definitive serotyping showed both to be S. indiana. The polyvalent antisera (pools and ) contained antibody against somatic,5, and 2. As noted previously,, 2 antigens reacted poorly by immunofluorescence with labeled,5,2 antibody. Thus, the low percentage ( to %) of somatic reactions observed with these isolates was to be expected. A polyvalent antibody pool containing,2 antibody would indicate more definitively the presence of group B strains. Nevertheless, the potential uses of immunofluorescence were indicated by this limited series for the rapid combined somatic grouping and serotyping of Salmonella. DISCUSSION In this study, a series of labeled H antisera prepared against many of the flagellar and somatic antigens of Salmonella were used to determine the parameters of specificity of immunofluorescence. H antisera were used because it was thought that combined flagellar and somatic staining of organisms in the same preparation was feasible, and that it would provide a means for overcoming the problem of crossreactions (9). In addition, the use of such sera permitted direct comparison of immunofluorescence with conventional agglutination reactions for an analysis of both and H antigens. The results demonstrated that flagellar antigens stained specifically. Somatic fluorescence was also specific, but its interpretation was not so straightforward as that of flagellar staining, principally because a given cell preparation stained with mixed intensity of brightness. Although the phenomenon of "percentage" reactions was observed at all levels of antigen-antibody relationship, from homologous to unrelated, it was more difficult to interpret when partially related systems were involved. Several possible reasons for this included incomplete washing of labeled antibody, partial dissociation of antibody from antigen, effects of formaldehyde on antigen, differences in classes of antibody reacting, presence of common or previously unrecognized minor Salmonella antigens, or differences in antigen distribution within a population. These possibilities will require further investigation. For the most part, however, somatic immunofluorescence was readily interpreted, particularly when the antigen-antibody systems were more closely related. The findings correlated well with these obtained by conventional agglutination procedures. The question arose as to whether the somatic titers of H antisera were a true Downloaded from on November 2, 28 by guest

10 86 CALDWELL, STULBERG, AND PETERSON J. BACTERIOL. TABLE 5. Presumptive serotyping of Salmonella with Spicer-Edwards polyvalent H labeled-globulins Spicer-Edwards polyvalent H labeledglobulins and antigens identified Probable antigens identified Specimen no. Pool H Pool 2 H Pool H Pool H H Most probable serotypes of each isolate,2,,5,,9, 2,9 a,bc, d,,g,i,2,6, 7,2 a,b,c,k,,2,5,6, a,d,e,,,, b,d,g,k, a,,,,7,8,2. h,k,z, 9,l,2, Gk r,y,z2g,2 Z,Z29 9, r,z.zls I a 9 ±,2,2 6,8 6,8 6,8 z z k r d S. ilndiana (,,2:z-,7)c S. worthingtont (,,2: I,w-z) S. poona (,22:z-,6) S. thompsoni (:k-,5)e S. blockley (6,8:k-,5) S. itifatitis (:r-,5)c S. heidelberg (,,5,2: r-,2) S. bovis-morbificans (6,8: r-,5) S. weltevredeni (,:r-z6) S. rubislaw (:r-e,n,x) S. muenichent (6,8:d-,2) S. typhosa (9,2,Vi: d--) S. manhattani (6,8:d-J,5)c S. grumpenisis (,2:d-,7) S. newport (6,8:-,2)c S. readinig (,5,2:-,5) S. anatum (,:-,6) S. san-diego (,2: -e,n,z,5) S. saint-paul (,,5,2: -,2) S. braeniderup (: -e,n,z,5) S. newingtonz (,5: -,6) - Approximate percentage of organisms with bright somatic peripheral fluorescence. bbright fluorescence of many flagella. c Confirmed by serotyping. measure of the actual titers, since heated antigens were reacted with antisera prepared against unheated H antigens. In addition to the correlations observed between agglutination and fluorescence, the validity of the measurements was further supported by the findings of repeated experiments where equivalent somatic agglutination titers were obtained with nonflagellated group B and group D antigens, regardless of whether or not they had been heated. The only apparent difference of H antisera was its lower titer and concomitant greater specificity for somatic antigen, when compared with antibody prepared in the usual manner, as indicated by the large number (52 of ) of instances where an expected partial relationship was not detected by agglutination. Although immunofluorescence generally paralleled agglutination, it was expected that quantitative differences would occur. Particularly Downloaded from on November 2, 28 by guest

11 VOL. 92, 966 IMMUNOFLUORESCENCE OF SALMONELLA 87 illustrative of this was the complete absorption of somatic immunofluorescence reactivity while substantial agglutination titers persisted. Qualitative differences were also noted when specific immunofluorescence was observed in the absence of detectable agglutination. Such results indicated the marked sensitivity of immunofluorescence for the recognition of minor antigenic relationships, which would have to be confirmed in each instance by specific absorption studies. Several technical modifications of the usual procedures employed for immunofluorescence staining of bacteria (,6) were developed which enhanced the feasibility of specific combined staining of and H antigens. These included: growth of organisms from either pure cultures or from fecal specimens in a suitable broth medium, as employed in Salmonella immunofluorescence studies by others (,); treatment of organisms with formaldehyde; and staining the organisms in suspension with labeled antibody. Collectively, these procedures aided in retaining flagellar integrity, eliminated much of the debris found in smear preparations, and provided for thorough mixing of labeled antiserum with the organisms, while enabling one to handle pathogenic material in a safe manner. These modifications were found to be applicable to the study of the specificity of immunofluorescence with organisms from pure cultures as well as with those from Salmonella specimens. Application of these immunofluorescence techniques to an epidemiological-diagnostic problem involving an outbreak due to a single Salmonella serotype (S. typhimurium) has been previously described in detail (8). The present study suggested that the potential for epidemiological and diagnostic use of the technique might be quite broad. Particularly pertinent was confirmation of the suitability of combined H and staining for interpretation of immunofluorescence, as shown by the results of salmonellosis surveys in mouse colonies. The large number of somatic cross-reactions observed were considered diagnostically negative in the absence of specific flagellar staining. This interpretation was supported by negative cultural findings. Similarly, a small number of flagellar reactions were encountered which were also considered negative in the absence of somatic staining. While cultural results and immunofluorescence results generally are not strictly comparable, in the present data there was agreement in an unusually high percentage of instances (97.6%). It should be noted that evidence of morphological comparability is an important auxillary criterion that can give significant information in any immunofluorescence staining procedure. The possibilities for presumptive serotyping of isolates were illustrated by the use of labeled Spicer-Edwards polyvalent antisera. The combined staining of H and antigens permitted presumptive identification of 7 serotypes, which suggested that more extensive study of this method would indicate the use of this kind of polyvalent labeled reagent for rapid screening procedures. An advantage of such procedures would be the speed with which presumptive grouping or typing of Salmonella might be accomplished by reference to somatic and flagellar reactions in the same preparation. ACKNOWLEDGMENTS We thank D. Freeman and Theresa Clogg for their technical assistance. This investigation was supported by Public Health Service contract PH -62-5, grant Al 859, and training grant GM-599 from the National Institutes of Health. LITERATURE CITED. CHERRY, W. B., AND M. D. MOODY Fluorescent-antibody techniques in diagnostic bacteriology. Bacteriol. Rev. 29: EDWARDS, P. R., AND W. H. EWING Identification of Enterobacteriaceae, 2nd ed. Burgess Publishing Co., Minneapolis.. GEORGALA, D. L., AND M. BOOTHROYD. 96. A rapid immunofluorescence technique for detecting salmonellae in raw meat. J. Hyg. 62: HAGLUND, J. R., J. C. AYRES, A. M. PATON, A. A. KRAFT, AND L. Y. QUINN. 96. Detection of Salmonella in eggs and egg products with fluorescent antibody. Appi. Microbiol. 2: KAUFFMANN, F. 96. Die bacteriologie der Salmonella-species. Munksgaard, Copenhagen. 6. PAGE, R. H., AND C. S. STULBERG Immunofluorescence in epidemiologic control of E. coli diarrhea. Am. J. Diseases Children : SIMPSON, W. F., AND C. S. STULBERG. 96. 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